Novel Cellular Compositions and Methods for Their Preparation

ABSTRACT

The present invention relates to novel cell (e.g., hepatocyte, etc.) compositions and methods for their preparation and use. In particular, the invention concerns methods of processing preparations of such cells so as to permit their repeated cryopreservation and thawing while retaining substantial viability. The invention also concerns preparations of cells (e.g., hepatocytes) that have been repeatedly cryopreserved and thawed.

This application is a continuation of U.S. patent application Ser. No.11/110,879, filed Apr. 21, 2005, which application is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel cell (e.g., hepatocyte, etc.)compositions and methods for their preparation and use. In particular,the invention concerns methods of processing preparations of such cellsso as to permit their repeated cryopreservation and thawing whileretaining substantial viability. The invention also concernspreparations of cells (e.g., hepatocytes) that have been repeatedlycryopreserved and thawed.

BACKGROUND OF THE INVENTION

Hepatocytes are parenchymal liver cells, and make up 60-80% of thecytoplasmic mass of the liver. Hepatocytes play a key role in thedetoxification, modification and excretion of exogenous and endogenoussubstances (Ponsoda, X. et al. (2004) “Drug Metabolism By Cultured HumanHepatocytes: How Far Are We From The In Vivo Reality?” Altern. Lab Anim.32(2):101-110). One of the detoxifying functions of hepatocytes is tomodify ammonia to urea for excretion. They are also important in proteinsynthesis and storage, in the transformation of carbohydrates and in thesynthesis of cholesterol, bile salts and phospholipids (Postic, C. etal. (2004) “Role Of The Liver In The Control Of Carbohydrate And LipidHomeostasis,” Diabetes Metab. 30(5):398-408). The hepatocyte is the onlycell in the body that manufactures albumin, fibrinogen, and theprothrombin group of clotting factors. It is the main site for thesynthesis of lipoproteins, ceruloplasmin, transferrin, andglycoproteins. Hepatocytes manufactures their own structural proteinsand intracellular enzymes. Hepatocytes are also important depots forvitamin B12 and iron.

Due to these attributes, isolated and cultured hepatocytes have becomevery attractive as models systems for the study of liver functions(Chesne, C. et al. (1993) “Viability And Function In Primary Culture OfAdult Hepatocytes From Various Animal Species And Human Beings AfterCryopreservation,” Hepatology 18(2):406-414; Guillouzo, A. et al. (1986)“Isolated and Cultured Hepatocytes,” Paris: les Editions INSERM andLondon: John Libbey Eurotext); Ponsoda. X. et al. (2004) “DrugMetabolism By Cultured Human Hepatocytes: How Far Are We From The InVivo Reality?” Altern. Lab Anim. 32(2):101-110; Gomez-Lechon, M. J. etal. (2004) “Human Hepatocytes In Primary Culture: The Choice ToInvestigate Drug Metabolism In Man,” Curr. Drug Metab. 5(5):443-462;Lemaigre, F. et al. (2004) “Liver Development Update: New Embryo Models,Cell Lineage Control, And Morphogenesis,” Curr Opin Genet Dev.14(5):582-590; Nanji, A. A. (2004) “Animal Models Of Nonalcoholic FattyLiver Disease And Steatohepatitis,” Clin Liver Dis. 8(3):559-574;Hewitt, N. J. et al. (2004) Cryopreserved Rat, Dog And MonkeyHepatocytes: Measurement Of Drug Metabolizing Enzymes In Suspensions AndCultures.” Hum Exp Toxicol. 23(6):307-316).

In addition to their use in liver models, hepatocytes have the potentialof being used to produce Bioartificial Livers (BALs) or in hepatocytetransplantation that can provide liver functions for individualssuffering from liver disease or liver failure. Bioartificial Livers(BALs) are described by Anand, A. C. (1996) “Bioartificial Livers: TheState Of The Art,” Trop Gastroenterol. 17(4):197-198, 202-211; Gan. J.H. et al. (2005) “Hybrid Artificial Liver Support System For TreatmentOf Severe Liver Failure,” World J. Gastroenterol. 11(6):890-894; Fukuda,J. et al. (2004) “Hepatocyte Organoid Culture In Elliptic Hollow FibersTo Develop A Hybrid Artificial Liver,” hit J Artif Organs.27(12):1091-1099; Meng, Q. et al. (2004) “Hepatocyte Culture InBioartificial Livers With Different Membrane Characteristics,”Biotechnol Lett. 26(18):1407-1412; Sekido, H. et al. (2004) “UsefulnessOf Artificial Liver Support For Pretransplant Patients With FulminantHepatic Failure,” Transplant Proc. 36(8):2355-2356; WO 03/105663A2,WO05/000376A2, and U.S. Pat. No. 6,759,245. Hepatocyte transplantationis described by Chan, C. et al. (2004) “Hepatic Tissue Engineering ForAdjunct And Temporary Liver Support: Critical Technologies,” LiverTranspl. 10(11):1331-1342; Lee, S. W. et al. (2004) “HepatocyteTransplantation: State Of The Art And Strategies For Overcoming ExistingHurdles,” Ann. Hepatol. 3(2):48-53; Horslen, S. P. (2004) “HepatocyteTransplantation,” Transplantation 77(10): 1481-1486; Burlina, A. B.(2004) “Hepatocyte Transplantation For Inborn Errors Of Metabolism,” J.Inherit. Metab. Dis. 27(3):373-83; and Fox, I. J. et al. (2004)“Hepatocyte Transplantation,” Am. J. Transplant. 4 Suppl. 6:7-13.

A limiting factor in the development of such model systems and to thedevelopment of Bioartificial Livers (BALs) has been the erratic sourceand limited availability of hepatocytes, especially human hepatocytes.Fresh hepatocytes are obtainable only from liver resections ornon-transplantable livers of multi-organ donors (Lloyd, T. D. R. et al.(2003) Cryopreservation Of Hepatocytes: A Review Of Current Methods ForBanking,” Cell and Tissue Culture Banking 4:3-15). The supply of suchtissue is inconsistent and often geographically inconvenient in light ofthe limited functional lifespan of liver tissue (Smrzova, J. et al.(2001) “Optimization Of Porcine Hepatocytes Cryopreservation ByComparison Of Viability And Enzymatic Activity Of Fresh AndCryopreserved Cells,” Acta Veterinaria Brunensis 70:141-147).

One approach to addressing this problem has involved the development ofhepatocyte storage conditions that allow hepatocytes to be maintainedover time with their cellular functions preserved. Cryopreservationmethods for the storage of hepatocytes have been developed to addressthis need (see, Lloyd, T. D. R. et al. (2003) Cryopreservation OfHepatocytes: A Review Of Current Methods For Banking,” Cell and TissueCulture Banking 4:3-15; Loretz, L. J. et al. (1989) “Optimization OfCryopreservation Procedures For Rat And Human Hepatocytes,” Xenobiotica.19(5):489-498; Shaddock, J. G. et al. (1993) “Cryopreservation AndLong-Term Storage Of Primary Rat Hepatocytes: Effects OnSubstrate-Specific Cytochrome P450-Dependent Activities And UnscheduledDNA Synthesis,” Cell Biol Toxicol. 9(4):345-357; Novicki, D. L. et al.(1982) “Cryopreservation Of structural proteins and intracellularenzymes. Hepatocytes are also important depots for vitamin B12 and iron.Isolated Rat Hepatocytes,” In Vitro. 18(4):393-399; Zaleski, J. et al.(1993) “Preservation Of The Rate And Profile Of Xenobiotic Metabolism InRat Hepatocytes Stored In Liquid Nitrogen,” Biochem Pharmacol.46(1):111-116). Typically, such measures comprise storage in liquidnitrogen (−196° C.) or in frozen nitrogen gas (−150° C.). The ability torecover viable thawed cells has been found to depend on multiple factorssuch as the rate of freezing, the concentration of hepatocytes, the typeof cryoprotectant employed, and the final cooling temperature. Cellconcentrations of 10⁶-10⁷ cells/ml have been typically employed. Theisolated hepatocytes are typically incubated in suspension for a period(e.g., 4-48 hours) to allow them to recover from the isolation process.Thereafter, a cryoprotectant (such as glycerol, DMSO,polyvinylpyrrolidone, or dextrin) is added, and the hepatocytes arefrozen. The art has developed various freezing procedures, all designedto minimize or prevent the occurrence of intracellular ice. The freezingrates typically vary from −0.05° C./min to −50° C./min (see, Lloyd, T.D. R. et al. (2003) Cryopreservation Of Hepatocytes: A Review Of CurrentMethods For Banking,” Cell and Tissue Culture Banking 4:3-15).

While the development of cryopreservation methods for the storage ofhepatocytes has significantly facilitated the availability of humanhepatocytes, cryopreservation has been found to cause a significantdecrease in cellular viability (e.g., 25-35%) (Dou, M. et al. (1992)“Thawed Human Hepatocytes In Primary Culture,” Cryobiology 29:454-469;Alexandre, E. et al. (2002) “Cryopreservation Of Adult Human HepatocytesObtained From Resected Liver Biopsies,” Cryobiology 44:103-113).Coundouris, J. A. et al. (1993) reported viability of 67% after 24hours, declining to 49% after 14 days (Coundouris, J. A. et al. (1993)“Cryopreservation Of Human Adult Hepatocytes For Use In Drug MetabolismAnd Toxicity Studies.” Xenobiotica. 23(12):1399-1409). Adams, R. M. etal. have reported that the viability of hepatocytes may be enhanced togreater than 90% using specialized cyropreservation fluids, however,only 16% of cells were found to be capable of replication (Adams, R. M.et al. (1995) “Effective _(—) Cryopreservation And Long-Terms Storage OfPrimary Human Hepatocytes With Recovery Of Viability, Differentiation,And Replicative Potential,” Cell Transplant. 4(6):579-586). Methods ofcryopreservation are disclosed in U.S. Pat. Nos. 5,795,711, 6,136,525,5,895,745; International Patent Publications WO04/009766, WO92/12722,WO/0153462, European Patent No. EP0834252B, and United States PatentApplications Publication Nos. US20020039786A1, US20030134418A1. The poorrecovery of cells when cryopreserved continues to limit the use ofhepatocytes in in vitro liver models.

A second major problem affecting the use of both fresh and cryopreservedhepatocytes is the variation of liver enzyme expression that is observedin tissue from different donors (Li. A. P. et al. (1999) “Present StatusOf The Application Of Cryopreserved Hepatocytes In The Evaluation OfXenobiotics: Consensus Of An International Expert Panel,” Chem BiolInteract. 121(1):117-123; Li, A. P. et al. (1999) “Cryopreserved HumanHepatocytes: Characterization Of Drug-Metabolizing Enzyme Activities AndApplications In Higher Throughput Screening Assays For Hepatotoxicity,Metabolic Stability, And Drug-Drug Interaction Potential,” Chem BiolInteract. 121(1):17-35; O'Brien, Z. Z. et al. (undated) “TheConstruction Of A Representative Human Cryopreserved Hepatocyte Pool ForMetabolism Study.” One solution to this sample variation involvespooling samples from different sources to produce a “composite”hepatocyte preparation having the characteristics of “average” livercells. However, the frequency of receipt of fresh tissue and the need tocryopreserve hepatocytes immediately after isolation has been cited aspreventing the preparation of hepatocyte pools. Thus, multiple companies(e.g., Xenotech, LLC; BD Biosciences) refrain from selling pooledhepatocytes thus forcing the end user to thaw and pool hepatocytes fromseveral different donors. This difficulty remains even though pooledcryopreserved human hepatocytes are a valid model for metabolic studies(Zhang, J. G. et al. (undated) “Validation Of Pooled Cryopreserved HumanHepatocytes As A Model for Metabolic Studies.”

Thus, despite all prior advances, a need remains for processes thatwould enable the availability of hepatocytes for medical research andother purposes. A need further exists for a stable and reproduciblesource of human hepatocytes. The present invention permits theproduction and availability of hepatocyte preparations that may berepeatedly cryopreserved and thawed without unacceptable loss ofviability. The invention thus permits multiple hepatocyte samples to bepooled to produce pooled hepatocyte preparations, especially pooledcryopreserved human hepatocyte preparations. Using such advance, pooledcryopreserved human hepatocytes are now commercially available from InVitro Technologies (Baltimore, Md.).

SUMMARY OF THE INVENTION

The present invention relates to novel cell (e.g., hepatocyte)compositions and methods for their preparation and use. In particular,the invention concerns methods of processing preparations of cells,especially hepatocytes, so as to permit their repeated cryopreservationand thawing while retaining substantial viability. The invention alsoconcerns preparations of cells (e.g., hepatocytes) that have beenrepeatedly cryopreserved and thawed.

In detail, the invention particularly concerns a multi-cryopreservedhepatocyte preparation comprising hepatocytes that have been frozen andthawed at least two times, wherein greater than 50% and more preferably70% or more of the hepatocytes of the preparation are viable.

The invention further concerns the embodiment of such amulti-cryopreserved hepatocyte preparation wherein the hepatocytes areselected from the group consisting of human hepatocytes, porcinehepatocytes, simian hepatocytes, canine hepatocytes, feline hepatocytes,bovine hepatocytes, equine hepatocytes, ovine hepatocytes and rodenthepatocytes.

The invention further concerns the embodiment of such amulti-cryopreserved hepatocyte preparation wherein the preparationcomprises a pooled preparation of hepatocytes of multiple sources, whichmay be of the same or different gender, race, or health state, or whichprovide the pooled preparation with a desired level of a metabolicactivity (especially wherein the metabolic activity is selected from thegroup consisting of COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST, PHEN andCZX).

The invention further concerns a method of producing a desiredpreparation of multi-cryopreserved hepatocytes, the hepatocytes beingcapable of being frozen and thawed at least two times, and in whichgreater than 50% and more preferably 70% or more of the hepatocytes ofthe preparation are viable, the method comprising:

(A) subjecting hepatocytes that have been frozen and thawed to densitygradient fractionation (especially PERCOLL® density centrifugation) toseparate viable hepatocytes from non-viable hepatocytes,

(B) recovering the separated viable hepatocytes, and

(C) cryopreserving the recovered viable hepatocytes to thereby form thedesired preparation of hepatocytes.

The invention further concerns the embodiment of such a method in whichthe hepatocytes are selected from the group consisting of humanhepatocytes, porcine hepatocytes, simian hepatocytes, caninehepatocytes, feline hepatocytes, bovine hepatocytes, equine hepatocytes,ovine hepatocytes and rodent hepatocytes.

The invention further concerns the embodiment of such a method in whichthe preparation comprises a pooled preparation of hepatocytes ofmultiple sources, which may be of the same or different gender, race, orhealth state, or which provide the pooled preparation with a desiredlevel of a metabolic activity (especially wherein the metabolic activityis selected from the group consisting of COUM, DEX, ECOD, 7-HCG, 7-HCS,MEPH, TEST, PHEN and CZX).

The invention also concerns a method of investigating in vitro drugmetabolism comprising incubating hepatocytes of a multi-cryopreservedhepatocyte preparation in the presence of a xenobiotic, and determiningthe metabolic fate of the xenobiotic, or the effect of the xenobiotic onthe hepatocytes or on an enzyme or metabolic activity thereof whereinthe hepatocytes have been frozen and thawed at least two times, andwherein greater than 50% and more preferably 70% or more of thehepatocytes of the preparation are viable.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to novel cell compositions and methods fortheir preparation and use. In particular, the invention concerns methodsof processing preparations of cells so as to permit their repeatedcryopreservation and thawing while retaining substantial viability. Themethods of the present invention are generally applicable to a widevariety of cell types, including hepatocytes, kidney cells, spleencells, thymus cells, bone marrow cells, stem cells, muscle cells(including cardiac muscle cells), endocrine cells (including pancreaticcells, adrenal cells, thyroid cells, etc.) epidermal cells, endodermalcells, etc. The methods of the present invention are illustrated belowwith respect to a preferred cell type: hepatocytes.

The invention also concerns preparations of cells, e.g., hepatocytes,that have been repeatedly cryopreserved and thawed to obtain a highviability preparation useful for a variety of experimental, diagnosticand therapeutic purposes. The present invention extends the ability ofhepatocytes to be cryopreserved and thawed for later use, so as topermit hepatocyte preparations to be repeatedly cryopreserved and thawedwithout an unacceptable loss of viability.

As used herein, the term “cell preparation” denotes a liquid or frozencomposition of cells from one or more sources (e.g., “hepatocytepreparation”, denotes a composition of liver cells from one or moresources). The sources may be primary cells that have been dissociatedfrom or isolated from tissue as by resection, biopsy, or from donororgans, or they may be secondary, immortalized or transformed cellcultures. The cells may be derived from any mammalian source, includinghuman, porcine, simian, canine, feline, bovine, equine, ovine or rodentsources. The use of human, porcine or rodent (especially rat) cells ispreferred. Preferably, greater than 50% and more preferably 70% or moreof the hepatocytes of such preparations will be viable.

As used herein, the term “multi-cryopreserved cell preparation” denotesa cell preparation that has been frozen and then thawed at least twotimes (e.g., a “multi-cryopreserved hepatocyte preparation” denotes ahepatocyte preparation that has been frozen and then thawed at least twotimes). Such preparations may have been frozen and thawed three, four,five, or more times.

The term “pooled preparation” denotes a cell (e.g., hepatocyte)preparation in which the cells (e.g., hepatocytes) are derived from two,three, four, five, or more different sources, such as different donors,biopsies, tissue resections from different tissue samples or differenttissue sources, or different primary, secondary, immortalized ortransformed cell (e.g., hepatocyte) cultures. The cells of such pooledpreparations may be randomly selected cell, or may have been selected toprovide the pooled preparation with a desired level of one or moremetabolic activities (such as for example, a preparation of hepatocyteshaving a desired level of COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST,PHEN and/or CZX activity), or a desired cell characteristic (such as,for example, a preparation of hepatocytes derived from sources of thesame gender, age, race (e.g., Caucasian, etc.), or health state (e.g.,hepatocytes of hepatitis virus-infected liver, hepatocytes of HIV-1infected liver, hepatocytes of healthy liver, hepatocytes of cigarettesmokers, hepatocytes of individuals suffering from cirrhosis of theliver, or from other diseases or conditions). For example, to obtain apooled hepatocyte preparation with minimal DEX activity, a pooledpreparation could be prepared from Lot. Nos. 067, CEK, ETR, PFM, VTA, orWWM (see, Table III).

In a preferred embodiment, illustrated with respect to hepatocyte cells,the practice of the invention comprises some or all of the followingsteps: the isolation of hepatocytes, a first cryopreservation of theisolated primary hepatocytes to obtain a first cryopreserved hepatocytepreparation, the thawing of the first cryopreserved hepatocytepreparation to obtain viable hepatocytes, and the reformulation of thethawed viable hepatocytes to permit their further storage and usethrough repeated cryopreservation and thawing to obtain viablehepatocytes.

The Isolation Of Hepatocytes

Any of a wide variety of methods may be employed or adapted to permitthe isolation of the primary hepatocytes used in the present invention.For 30 example, suitable techniques for the isolation of hepatocytes areoutlined in Morsiani et al, (1995) “Automated Liver Cell ProcessingFacilitates Large Scale Isolation And Purification Of PorcineHepatocytes,” ASAIO Journal 41:155-161 and in Seglen, P. O. (1976)“Preparation Of Isolated Rat Liver Cells,” Meth. Cell Biol. 13:29-83).Specific reference is made to the two-step collagenase digestionprocedure described in Li, A. P. et al. (1992) “Isolation And CulturingOf Hepatocytes From Human Liver,” J. Tissue Cult. Meth. 14:139-146.

The hepatocytes may be cultured in any suitable hepatocyte culturemedium. By way of illustration and not limitation mention may be made ofthe following culture media Chee's Essential Media (Hamilton, G. A. etal. (2001) “Effects Of Medium Composition On The Morphology And FunctionOf Rat Hepatocytes Cultured As Spheroids And Monolayers,” In Vitro CellDev Biol Anim. 37(10):656-667; Zurlo, J. et al. (1996) “CharacterizationOf A Primary Hepatocyte Culture System For Toxicological Studies,” InVitro Cell Dev Biol Anim. 32(4):211-220; Arterburn, L. M. et al. (1995)“A Morphological Study Of Differentiated Hepatocytes In Vitro,”Hepatology 22(1):175-187), Modified Eagle Medium (or Dulbecco's ModifiedEagle Medium) (Arikura, J. et al. (2002) “UW Solution: A Promising ToolFor Cryopreservation Of Primarily Isolated Rat Hepatocytes,” JHepatobiliary Pancreat Surg. 9(6):742-749; Washizu, J. et al. (2000)“Amino Acid Supplementation Improves Cell-Specific Functions Of The RatHepatocytes Exposed To Human Plasma,” Tissue Eng. 6(5):497-504; Iwata,H. et al. (1999) “In Vitro Evaluation Of Metabolic Functions Of ABioartificial Liver,” ASAID J. 45(4):299-306; Stutenkemper, R et al.(1992) “The Hepatocyte-Specific Phenotype Of Murine Liver CellsCorrelates With High Expression Of Connexin32 And Connexin26 But VeryLow Expression Of Connexin43,” Exp Cell Res. 201(1):43-54), Leibowitzmedium (Coundouris, J. A. et al. (1993) “Cryopreservation Of Human AdultHepatocytes For Use In Drug Metabolism And Toxicity Studies,”Xenobiotics: 23(12):1399-1409), Waymouth (Vind, C. et al. (1992)“Regulation By Growth Hormone And Glucocorticoid Of TestosteroneMetabolism In Long-Term Cultures Of Hepatocytes From Male And FemaleRats,” Biochem Pharmacol. 44(8):1523-1528; Nemoto, N. et al. (1991)“Proline Is Required For Transcriptional Control Of The AromaticHydrocarbon Inducible P(1) 450 Gene In C57BL16 Mouse Monolayer-CulturedHepatocytes,” Jpn J Cancer Res. 82(8):901-908; Dich, J. et al. (1988)“Long-Term Culture Of Hepatocytes: Effect Of Hormones On EnzymeActivities And Metabolic Capacity,” Hepatology. 8(1):39-45; Goethals, F.et al. (1984) “Critical Biochemical Functions Of Isolated Hepatocytes AsSensitive Indicators Of Chemical Toxicity,” Fundam Appl Toxicol. 4 (3 Pt1):441-450), Kreb's medium (House, J. D. (2001) “Threonine Metabolism InIsolated Rat Hepatocytes,” Am J Physiol Endocrinol Metab.281(6):E1300-1307; Irvine, F. et al. (1993) “Extracellular CalciumModulates Insulin's Action On Enzymes Controlling Cyclic AMP MetabolismIn Intact Hepatocytes,” Biochem J. 293 (Pt 1):249-253; Marsh, D. C. etal. (1991) “Hypothermic Preservation Of Hepatocytes. III. Effects OfResuspension Media On Viability After Up To 7 Days Of Storage,”Hepatology 13(3):500-508), etc.

In a preferred embodiment, hepatocytes are cryopreserved in a mediumcontaining approximately 10% DMSO and approximately 90% fetal bovineserum (Loretz, L. J. et al. (1989) “Optimization Of CryopreservationProcedures For Rat And Human Hepatocytes,” Xenobiotics 19:489-498;Ruegg, C. E. et al. (1997) “Cytochrome-P450 Induction and ConjugatedMetabolism In Primary Human Hepatocytes After Cryopreservation,” InVitro Toxicol. 10:217-222).

The viability of the isolated hepatocytes may be determined using any ofa variety or methods. Preferable, such viability will be determinedusing the Trypan blue exclusion method (see, e.g., Berry, M. N. et al.(1992) “Techniques For Pharmacological And Toxicological Studies WithIsolated Hepatocyte Suspensions,” Life Sci. 51(1):I-16). Thus thephrases “viable hepatocytes” or “percent viability”, as used herein,refers to hepatocyte viability as assessed using the method of TrypanBlue exclusion.

Cryopreservation of the Isolated Primary Hepatocytes

The hepatocytes of the present invention are preferably cryopreservedusing liquid nitrogen, and most preferably within 36 hours of theirisolation. Considerations for the cryopreservation of human hepatocytesare discussed in Lloyd, T. D. R. et al. (2003) Cryopreservation OfHepatocytes: A Review Of Current Methods For Banking,” Cell and TissueCulture Banking 4:3-15. Suitable procedures for the cryopreservation ofhepatocytes may also be found in the following documents: Adams, R. M.et al. (1995) “Effective Cryopreservation And Long-Term Storage OfPrimary Human Hepatocytes With Recovery Of Viability, Differentiation,And Replicative Potential,” Cell Transplant. 4(6):579-586; Chesne, C. etal. (1993) “Viability And Function In Primary Culture Of AdultHepatocytes From Various Animal Species And Human Beings AfterCryopreservation,” Hepatology 18(2):406-414; Coundouris, J. A. et al.(1993) “Cryopreservation Of Human Adult Hepatocytes For Use In DrugMetabolism And Toxicity Studies,” Xenobiotica. 23(12):1399-1409; Hewitt,N. J. et al. (2004) Cryopreserved Rat Dog And Monkey Hepatocytes:Measurement Of Drug Metabolizing Enzymes In Suspensions And Cultures,”Hum Exp Toxicol. 23(6):307-316; Novicki, D. L. et al. (1982)“Cryopreservation Of Isolated Rat Hepatocytes,” In Vitro. 18(4):393-399;Shaddock, J. G. et al. (1993) “Cryopreservation And Long-Term Storage OfPrimary Rat Hepatocytes: Effects On Substrate-Specific CytochromeP450-Dependent Activities And Unscheduled DNA Synthesis,” Cell BiolToxicol.9(4):345-357; Zaleski, J. et al. (1993) “Preservation Of TheRate And Profile Of Xenobiotic Metabolism In Rat Hepatocytes Stored InLiquid Nitrogen,” Biochem Pharmacol. 46(1):111-116.

Preferably, isolated hepatocytes are suspended in a cryoprotectivemedium, and the suspended cells are dispensed into freezer-safecontainers. A cryoprotective medium typically comprises a hepatocyteculture medium that contains at least one cryoprotectant that minimizesthe deleterious effects of cryopreservation such as the formation ofintracellular ice during freezing. By way of illustration and notlimitation, the following commonly used cryoprotectants are listed:dimethylsulfoxide (DMSD), polyethylene glycol, amino acids, propanediol,and glycerol. A preferred cryoprotectant of the present invention isDMSD. Suitable cryoprotectants and methods for their use in hepatocytecryopreservation can be found, for example, in: Lore, L. J. et al.(1989) “Optimization Of Cryopreservation Procedures For Rat And HumanHepatocytes,” Xenobiotica. 19(5):489-498; Chesne, C. et al. (1993)“Viability And Function In Primary Culture Of Adult Hepatocytes FromVarious Animal Species And Human Beings After Cryopreservation,”Hepatology 18(2):406-414; Diener, B. et al. (1993) “A Method For TheCryopreservation Of Liver Parenchymal Cells For Studies Of Xenobiotics,”Cryobiology 30(2):116-127; Lawrence, J. N. et al. (1991) “Development OfAn Optimal Method For The Cryopreservation Of Hepatocytes And TheirSubsequent Monolayer Culture. Toxicology In Vitro,” 5(1):39-51; Houle,R. et al. (2003) “Retention of Transporter Activities in Cryopreserved,Isolated Rat Hepatocytes,” Drug Metab. Disposit. 31(4):447-451; Silva,J. M. et al. (1999) “Induction Of Cytochrome-P450 In Cryopreserved RatAnd Human Hepatocytes,” Chem-Biol Interact 121:49-63.

The isolated hepatocytes are preferably suspended in a cryoprotectivemedium in preparation for freezing. The suspended cells are preferablydispensed into freezer resistant containers at a cell density of fromabout 105 cells/ml to about 4×10′ cells/ml. Preferred freezing volumesrange from 0.1-10.0 ml. The preferred freezing volume is 1.0 ml.

The dispensed hepatocytes are then preferably cryopreserved using acontrolled rate freezing process, most preferably at a freezing rate ofbetween about −1° C./min to about −25° C./min until a final temperatureof about −90° C. is reached. During the initial phase of thecryopreservation process, seeding may be employed to induce controlledcrystallization or ice formation in cell suspensions that have alreadybeen cooled to below the freezing point of the culture medium. Suchseeding serves to minimize ice formation-related damage and thereforemay be beneficial to cell viability. Suitable seeding methods includeinserting a cold metal rod into the freezing containers, and introducinga blast of liquid nitrogen into the freezing containers.

Once the desired final temperature has been reached, the frozen cellsamples may be transferred to liquid nitrogen freezers for prolongedstorage. The frozen samples may be stored in either the liquid nitrogenphase or the gas phase of liquid nitrogen. Preferably storage isaccomplished in the gas phase of liquid nitrogen. The frozen samples maybe stored in this manner for days, months, or years, with the length ofstorage in the gas phase of liquid nitrogen having little effect on thepost-thaw viability and function.

The Thawing of Cryopreserved Hepatocytes

Frozen samples may be thawed for further processing by removing themfrom the presence of liquid nitrogen or liquid nitrogen vapor. Frozensamples are preferably thawed by placing the samples immediately into aprewarmed water bath having a temperature of between about 37° C. toabout 42° C. Preferably, cells are thawed to at least the stage in whichice chunks can be dislodged when the sample container is inverted. Thethawed cells are then preferably rapidly processed to remove the cellsfrom contact with DMSO, for example by PERCOLL® (colloidal silicaparticles of 15-30 nm diameter (23% w/w in water) which have been coatedwith polyvinylpyrrolidone (PVP)) gradient centrifugation (as describedbelow) or by sequential washings.

In a preferred embodiment, the cells are thawed into CompleteINVITROGRO™ CP medium (In Vitro Technologies, Baltimore, Md.; Roymans.U. et al. (2004) “Determination Of Cytochrome P450 IA2 And CytochromeP4503a4 Induction In Cryopreserved Human Hepatocytes,” Biochem.Pharmacol. 67(3):427-437) (hepatocyte plating medium, which containswater, Dulbecco's Modified Eagle Medium, sodium bicarbonate, HEPES,fructose, bovine serum albumin, sodium hydroxide, MEM non-essentialamino acids, insulin, hydrocortisone, and newborn calf serum). Themedium is prepared by thawing TORPEDO™ Antibiotic Mix (In VitroTechnologies, Baltimore, Md.) (a mixture of antibiotics selected toinhibit bacterial growth in hepatocyte cell cultures that containspenicillin, streptomycin, gentamicin, amikacin and fungizone) to 37° C.in a water bath until thawed, and then removed from the water bath. 1.0ml of TORPEDO™ Antibiotic Mix is then mixed with 45 ml INVITROGRO™ CPmedium. Following the addition of TORPEDO™ Antibiotic Mix, the shelflife for the complete medium is 7 days. When thawing a single vial, theINVITROGRO™ CP medium is prewarmed to approximately 37° C. 5 ml ofwarmed INVITROGRO™ CP medium is added to a sterile 50 ml conical tube.The vial of frozen hepatocytes is carefully removed from the freezer. Ifthe vial was stored in the liquid phase, its cap is carefully removed,any liquid nitrogen present in the vial is decanted, and the cap isreclosed before placing the vial into the water bath. It is preferred tothen immediately immerse the vial into a 37° C. water bath, and to shakethe vial gently until the ice is entirely melted, but no longer than ittakes to completely thaw the vial. It may be helpful to remove anylabels from the vial so that it will be easier to view the vialcontents. The thawed contents are then emptied into the pre-warmedINVITROGRO™ CP medium. 1.0 ml of pre-warmed INVITROGRO™ CP medium thenis added to each vial to resuspend any remaining cells. The contents ofthe vial are then decanted or pipetted into the hepatocyte suspension.The hepatocytes are preferably resuspended by gently inverting thereceiving container (e.g., vial, test rube, etc.) several (e.g., three)times.

When thawing multiple vials, it is preferred that all of the vials bethawed in the water bath simultaneously. As before, the medium(preferably, INVITROGRO™ CP medium) should be warmed to 37° C. It isdesirable to ensure that there is enough medium to permit 5 ml ofpre-warmed INVITROGRO™ CP medium to he used for each vial ofcryopreserved hepatocytes. After vials have thawed, their caps should bequickly removed and their contents poured into a sterile tube or beakerthat contains at least 5 ml of pre-warmed INVITROGRO™ CP medium per vialthawed. For example, 25 ml of media is preferably employed for 5 vialsin a container that can hold a volume of 50 ml.

If desired, the total cell count and the number of viable cells may bedetermined using the Trypan Blue exclusion method. Cells may he dilutedto 0.70×10⁶ viable cells/ml with INVITROGRO™ CP medium.

The Reformulation of Thawed Hepatocytes to Permit FurtherCryopreservation and Thawing

One aspect of the present invention concerns the ability to reformulatethe thawed cells so that they may be refrozen and rethawed on one ormore subsequent occasions. Such multi-cryopreserved hepatocytepreparations have multiple uses. They may be used in bioartificiallivers, liver cell transplants, liver assist devices, hepatocytetransplantations, and in vitro applications. In particular,multi-cryopreserved hepatocyte preparations may be used in in vitro drugmetabolism studies (for example, in identifying hepatocytes with uniquecharacteristics (e.g., metabolic polymorphisms, genetic polymorphisms,etc.), in studies on the metabolic fate of the xenobiotic and studies onthe affect of the xenobiotic in altering the drug-metabolizing enzymeprofile of the hepatocytes, in inhibition studies to determine the IC50of xenobiotics on liver enzymes and functions (e.g. cholesterolmetabolism), in gene induction studies with xenobiotics, in proteininduction studies with xenobiotics, in toxicity assessment ofxenobiotics on hepatocytes, transport studies with xenobiotics (e.g.studies on P-glycoprotein transport systems, organic ion transporters,organic cation transporters, etc.), in metabolic clearance studies withxenobiotics, and in efficacy assays (e.g. lipoprotein processing,gluconeogenesis, protein secretion etc.). Multi-cryopreserved hepatocytepreparations may also be used to study or propagate hepatitis virusesand other infectious viruses and agents. Recovered cells may bereformulated for use in DNA, mRNA or proteomic studies or in studies ofmetabolic polymorphisms. Multi-cryopreserved hepatocyte preparations mayalso be used in metabolic clearance studies and efficacy assays (e.g.,lipoprotein processing, gluconeogenesis, protein secretion, etc.). Cellsmay be reformulated for use in seeding bioreactors for large scaleincubations or as models for gene regulation via micro RNA, or for usein combination systems with other cell types (e.g. non-parenchymal cellsfrom liver or cells from other sources, e.g. Caco-2 cells).

In a preferred embodiment of the invention, such reformulation comprisesseparating viable and non-viable cells prior to a subsequent refreezing.Density gradient centrifugation is preferably employed for this purpose.For example, a 30% PERCOLL® gradient centrifugation procedure may beemployed (Madan. A. et al. (1999) “Effect of Cryopreservation onCytochrome P-450 Enzyme Induction in Cultured Rat Hepatocytes, DrugMetab. Dispos. 27(3):327-335; Sun, E. L. et al. (1990) “CryopreservationOf Cynomologus Monkey (Macaca fascicularis) Hepatocytes For SubsequentCulture And Protein Synthesis Studies,” In Vitro Cell Development andBiology 25:147-150; Lawrence, J. N. et al. (1991) “Development Of AnOptimal Method For The Cryopreservation Of Hepatocytes And TheirSubsequent Monolayer Culture,” Toxicology In Vitro, 5(1):39-51; Dou, M.et al. (1992) “Thawed Human Hepatocytes In Primary Culture,” Cryobiology29(4):454-469; Utesch, D. et al. (1992) “Characterization OfCryopreserved Rat Liver Parenchymal Cells By Metabolism Of DiagnosticSubstrates And Activities Of Related Enzymes,” Biochemical Pharmacology44:309-315). For example, the thawed cells may be resuspended in aprewarmed (approximately 37° C.) 30% PERCOLL® isotonic fractionationbuffer and then centrifuged at 100×g at room temperature for twentyminutes to pellet viable cells. The supernatant is discarded and thecells are resuspended in media for a subsequent cryopreservation stepdirectly or for further processing prior to cryopreservation.

Cryopreserved preparations that result from the freezing of a previouslyfrozen-thawed preparation will preferably have a post-thaw cellviability of greater than 50% and more preferably 70% or more. Such highviabilities enable the present invention to accomplish the repeatedfreezing and thawing of hepatocytes without unacceptable losses of cellsor the need for ever greater samples sources.

Pooled Hepatocyte Preparations

The capacity of the present invention to enable the repeated freezingand thawing of hepatocytes additionally facilitates the production ofpooled hepatocyte preparations, especially pooled human hepatocytepreparations. As discussed above, individual liver samples yieldhepatocytes having differing metabolic capabilities. In order tofacilitate the reproducible use or study of hepatocytes, it is desirableto minimize hepatocyte differences attributable to such sample variationby pooling hepatocytes from different sources to obtain a composite or“average” hepatocyte preparation. Such composite hepatocyte preparationsmay thus be formulated so as to provide a preparation having themetabolic activities of an “average” hepatocyte sample or a preparationwhose hepatocyte enzyme functions approximate the hepatocyte enzymefunctions of freshly isolated hepatocytes. Such metabolic activities mayinclude, for example, some or all of the following enzymatic activities:coumarin 7-hydroxylase (COUM), dextromethorphan O-demethylase (DEX),7-ethoxycoumarin O-deethylase (ECOD), activities responsible for thephase II metabolism of 7-hydroxycoumarin (7-HCG and 7-HCS), mephenyloin4-hydroxylase (MEPH), testosterone 6(B)-hydroxylase (TEST), tolbutamide4-hydroxylase (TOLB), phenacetin O-deethylase (PHEN), or chlorzoxazone6-hydroxylase (CZX). The substrates, methods of measurements and assayunits for assays of such metabolic activities are provided in Table 1.

TABLE I Hepatocyte Metabolic Activities Abbreviation Substrate/AssayMethod of Measurement Units 7-HCG 7- Phase II metabolism of 7-Pmol/min/10⁶ cells hydroxycoumarin hydroxycoumarin glucuronide 7-HCS 7-Phase II metabolism Pmol/min/10⁶ culls hydroxycoumarin of7-hydroxycoumarin sulfate NAT1 p-aminobenzoic acid N-acetylation of p-nmol/mg/min aminobenzoic acid NAT2 Sulfamethazine N-acetylation ofnmol/mg/min Sulfamethazine VBTY viability Trypan Blue ™ percentageExclusion AP Alkaline Phosphatase Sigma kit units/mg protein GGTgamma-glutamyl GGT stain positive transpeptidase UGT1 7-hydroxycoumarinPhase I1 metabolism  169 pmol/mg/min of 7-hydroxycoumarin P450cytochrome p450 carbon monoxide Not determined for content differencespectrum cryo hepatocytes nmol/mg protein CZX chlorzoxazonechlorzoxazone 6- 31.1 pmol/mg/min* hydroxylation COUM coumarin coumarin7- 50.0 pmol/mg/min* hydroxylation DEX dextromethorphan dextromethorphanO- 21.4 pmol/mg/min* demethylation MEPH mephenytoin mephenytoin 4- 24.1pmol/mg/min* hydroxylation PHEN phenacetin phenacetin O- 28.9pmol/mg/min* deethylation TEST testosterone testosterone 6(beta)- 96.8pmol/mg/min* hydroxylation TOLB tolbutamide tolbutamide 4- 30.6pmol/mg/min* hydroxylation PROT protein content Pierce protein kit Notdetermined for cryo hepatocytes mg/mL ECOD ethoxycoumarin7-ethoxycoumarin O- 37.3 pmol/mg/min* demethylation

For example, preferred preparations of pooled hepatocytes will yieldassay values within the ranges identified in Table II. Alternatively,the hepatocytes samples used to form the pooled preparation may beselected so as to maximize, minimize, or emphasize certain hepatocytefunctions over other functions so as to yield a pooled preparation thatexhibits a user desired profile of liver cell function(s).

The pooled hepatocyte preparations of the present invention may comprisehepatocytes obtained from the same source at differing times, or fromtwo or more different sources. Preferably, pooled hepatocytepreparations will result from the pooling of hepatocytes obtained fromthree, four, five, six or more different sources.

Most preferably, the pooled hepatocyte preparations of the inventionwill comprise at least one population of hepatocytes that werecryopreserved prior to pooling. For example, a pooled hepatocytepreparation may comprise one or more hepatocyte specimens that werecryopreserved prior to pooling with one or more freshly isolatedhepatocyte specimens. Alternatively, a pooled hepatocyte preparation maycomprise only hepatocyte specimens that were previously cryopreserved.Table II provides the normal range (i.e., the range between AssayMinimum and Assay Maximum for each Assay). Table II values are deriveddata of the last 150+ lots of human cryopreserved hepatocytes.

TABLE II Hepatocyte Assays Normal Range Assay (pmol/min/10⁶ cells)coumarin 7-hydroxylation 1 to 154 Dextromethorphan O-demethylation 0.5to 96   7-ethoxycoumarin O-deethylation 1 to 154 Phase I metabolism of7-hydroxycoumarin 2 to 545 Phase II metabolism of 7-hydroxycoumarin 0 to110 mephenytoin 4-hydroxylation 0.2 to 442   testosterone6(beta)-hydroxylation 2 to 675 tolbutamide 4-hydroxylation 1.8 to 82  phenacetin O-deethylation 1 to 125 chlorzoxazone 6-hydroxylation 2 to215

In certain embodiments of the invention, hepatocyte preparations willhave assay values in the above stated ranges for at least three of, andpreferably for at least four of, still more preferably for at least sixof, and most preferably for at least eight of the following assays: theCOUM assay; the DEX assay; the ECOD assay; the 7-HCG assay; the 7-HCSassay; the MEPH assay; the TEST assay; the TOLB assay; the PHEN assay;the CZX assay.

If desired, the cryopreserved hepatocytes may be plated on tocollagen-coated tissue culture plates, or tissue culture plates coatedwith other extracellular matrix proteins including but not limited tolaminin, fibronectin, entactin, poly-L-lysine, gelatin, or anycombination thereof. Preferably, this is accomplished by diluting anappropriate volume (e.g., 0.2 ml to 2.5 ml) of diluted cells (e.g.,cells having a concentration of approximately 0.7×106 cells/ml) onto theplates. For plating on a 96-well microliter plate, it is desirable tofurther dilute the cell suspension to a concentration of 0.35×106cells/ml with INVITROGRO™ Medium, and to add 100 μl of the cellsuspension to each well. It is preferred to even distribute the cells inthe wells. This can be accomplished by gently shaking the plates in aback-and-forth and side-to-side manner; the use of a circular motionwill cause the cells to evenly pool in the center of the wells. Humanhepatocytes handled in this manner will attach to the plates in 2-4hours, however, if minimal handling is desired, the cells can be allowedto attach overnight.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

Example 1 Refreezing of Thawed Hepatocyte Preparations

Cryopreserved hepatocytes are thawed and refrozen as indicated below.

Materials: 30 cc syringe, two couplers, PERCOLL® in a 1 L bag,INVITROGRO™ CP medium in a 2 L bag, 1-2 L autoclaved beaker, heater,waterbath, centrifuge tube rack.

Procedure:

1) Set up a recirculating waterbath heater at 37-42° C.

2) Add approximately 200-400 mls of INVITROGRO™ CP medium to a 1-2 Lbeaker. Equip Biological Safety Cabinet with a manual pipet or a liquidhandling robot.

3) Remove approximately 50 cryovials from dewar receptacle and quicklyplace them in 2 test tube racks. When possible, space vials apart.

4) Submerge solid cell suspensions into heated waterbath until the icechunks can be dislodged when the vial is inverted.

Pour cell suspension from each vial into the beaker. Add 1 ml ofINVITROGRO™ CP medium from the small beaker into each vial to rinse andpour contents into the beaker. Transfer thawed cell suspension into a 1L sterile bag.

6) Attach bag, INVITROGRO™ CP medium in a 2 L bag and PERCOLL® bagged at30% onto COBE® automated cell processor (a centrifugal systemincorporating a flexible membrane that allows the removal of fluidswhile spinning through use of a rotating seal, hydraulic pump andflexible membrane) and process according to standard practices.

7) Perform a cell count.

8) Cryopreserve cell suspension.

PERCOLL®/REDIGRAD™ (Amersham Biosciences) is employed for the PERCOLL®density centrifugation. PERCOLL® is composed of colloidal silica coatedwith polyvinylpyrrolidone (PVP). The REDIGRAD™ formulation is alsocomposed of colloidal silica but is covalently coated with silane. Thesecoatings are thought to render the material non-toxic and ideal for usewith biological materials. Both particles have a density of 1.13 g/ml.Centrifugation of samples in the presence of PERCOLL®/REDIGRAD™ resultsin the spontaneous formation of a density gradient due to theheterogeneity of particle sizes in the medium.

PERCOLL®/REDIGRAD™ is best used in balanced salt solutions, such asphysiological saline (0.15M NaCl), although 0.25 M sucrose may beemployed. The addition of 9 parts (v/v) of PERCOLL®/REDIGRAD™ to 1 part(v/v) of either 1.5 M NaCl, 10× concentrated cell culture medium, or 2.5M sucrose will result in a solution adjusted to about 340 mOs/kg H₂O,Solutions of different osmotic pressure can be produced by adjusting therelative volumes of PERCOLL®/REDIGRAD™ and salt or sucrose solution.(Vincent, R. et al. (1984) “Adjustment Of The Osmolality Of Percoll ForThe Isopycnic Separation Of Cells And Cell Organelles,” Anal. Biochem.141(2):322-328). The final adjustment to the required osmolality can becarried out by the addition of salts or distilled water. Concentrationsother than 10× physiological saline may also be used satisfactorily.

PERCOLL®/REDIGRAD™ will form self-generated gradients by centrifugationin fixed-angle rotor heads after 15 minutes. Hepatocytes can beseparated by centrifugation at 50-100 g_(av) in fixed-angle or swingingbucket rotor heads after 10-30 minutes.

Example 2 Variation of Primary Hepatocyte Samples

To illustrate the sample-to-sample variation of different sources ofindividual (unpooled) hepatocytes, hepatocytes are isolated from 82different donors and analyzed for cell viability and enzyme function.The following metabolic activities are evaluated: COUM, DEX, ECOD,7-HCG, 7-HCS, MEPH, TEST, TOLB, PHEN, and CZX. The results are shown inTable III.

TABLE III Variation of Hepatocyte Samples 7- 7- Lot No. Sex % V COUM DEXECOD HCG HCS MEPH TEST TOLB PHEN CZX 067 M 62% 67 1 70 231 24 2 44 35 2724 086 F 74% 51 23 10 50 9 1 38 13 BQL 18 089 F 77% 25 21 8 23 6 1 11 13BQL 29 090 F 74% 30 25 7 13 BQL 2 19 15 BQL 16 091 F 73% 13 29 66 44 1012 252 36 27 36 094 F 67% 41 12 37 24 4 21 126 40 19 87 099 F 86% 21 157 4 BQL 1 60 10 BQL 22 104 M 81% 63 21 44 247 25 2 58 37 8 20 105 M 67%59 15 24 38 14 1 29 27 12 27 110 F 77% 45 24 35 23 4 6 206 11 54 36 111F 71% 4 10 9 2 3 3 147 2 19 19 114 F 75% 39 23 21 10 5 5 59 TBD 3 45 122M 79% 26 30 29 80 5 1 42 23 4 25 129 F 90% 4 24 27 67 18 1 16 33 10 51ACU F 81% 53 8 25 74 18 8 80 16 4 23 AIT F 83% 45 29 13 118 14 BQL 82 1511 7 AOK M 73% 60 21 58 283 64 5 86 30 24 21 ATR M 73% 7 11 1 39 11 BQL11 8 3 4 AVF M 70% 59 13 50 210 29 BQL 54 37 11 24 BTP M 88% 66 29 36214 25 3 50 45 11 12 CEC M 86% 47 26 17 105 27 21 32 57 38 36 CEK F 80%55 2 39 141 5 16 302 41 7 16 CHD F 77% 30 14 53 471 42 4 28 23 13 26 CPNM 81% 28 6 40 100 13 2 168 14 37 21 ECM M 85% 8 11 10 55 18 6 81 18 21 9EFA M 69% 9 9 35 47 5 18 66 12 47 67 EHI F 90% 88 3 56 291 45 1 248 2145 43 EJR F 75% 89 14 32 288 43 8 62 41 1 41 ENR M 73% 69 28 27 124 31107 77 36 38 32 EOB M 88% 14 11 18 65 9 31 49 14 21 35 ETR F 88% 30 1 3413 13 7 13 13 37 69 EVY M 80% 2 20 23 218 77 33 24 25 17 38 FNL M 85% 1727 62 282 32 6 6 50 14 24 FRW M 75% 46 22 16 106 17 48 25 54 44 8 GBE F77% 5 49 31 165 20 2 16 19 5 54 GNG F 74% 57 17 33 54 8 5 95 22 16 22GTV F 71% 32 6 8 47 7 BQL 40 28 16 11 GUY M 92% 65 12 11 73 12 20 90 135 8 HHG M 83% 2 8 14 251 29 BQL 28 12 4 40 HRU M 90% 43 28 39 175 15 469 40 57 44 ICJ M 74% 134 20 60 287 17 BQL 129 82 28 7 IEM M 88% 34 1723 129 34 72 48 23 19 34 IHR F 76% 17 43 8 84 9 9 95 46 41 7 LID M 86%36 31 50 307 54 1 142 49 21 51 IRX F 73% 57 5 40 172 24 12 113 43 6 18JUL M 82% 7 11 3 41 9 7 23 12 2 3 KK5 M 83% 1 8 27 319 38 BQL 61 17 1742 KPT F 83% 9 12 32 248 30 55 65 26 56 32 KRJ F 76% 6 40 76 359 37 1 1161 23 20 KRM F 78% 126 36 55 83 17 103 98 46 74 44 K5E M 73% 65 27 52206 74 21 123 42 93 16 KZO F 82% 38 16 38 262 8 6 75 32 30 20 LAE M 76%58 15 50 294 22 14 67 63 125 21 MOF F 91% 79 17 29 10 12 2 85 5 7 46 MRSM 72% 119 21 110 450 50 2 675 54 68 28 MTR F 69% 2 33 23 218 3 5 38 6739 8 MYO F 94% 40 24 9 24 BQL BQL 12 7 BQL 11 NPX F 79% 36 32 13 130 615 76 25 20 10 NQT M 85% 76 12 39 80 23 2 151 14 20 34 OAU F 81% 47 2624 86 8 6 85 46 53 13 OZL M 76% 16 15 61 300 109 3 165 29 17 43 PFM F87% 21 1 11 67 10 3 116 8 15 33 PXK M 80% 86 35 63 433 78 2 109 62 32 60QWG F 77% 16 32 29 300 21 9 50 10 BQL 15 REL F 77% 40 20 15 109 9 65 10033 75 10 RFA F 78% 130 42 49 444 52 6 195 30 28 17 RKB F 95% 42 16 16100 8 3 36 17 20 19 RML F 76% BQL 6 45 129 31 14 152 24 42 29 RNG F 91%119 14 97 298 27 177 207 34 71 41 ROE F 82% 73 24 36 302 37 2 55 17 2 51SEO F 72% 36 25 18 106 9 66 102 50 81 11 SQJ F 74% 115 12 100 285 19 175210 30 81 42 SRA M 79% 50 6 71 409 84 10 23 28 18 44 TPZ F 83% 120 13101 301 26 171 204 31 82 41 TSR F 62% 47 66 58 175 20 BQL 6 77 16 34 VCMM 82% 42 28 79 415 110 0.2 94 16 4 215 VEN F 70% 79 69 89 328 53 2 32 5848 81 VTA M 78% 32 1 25 84 12 5 120 21 40 15 WWM M 84% 42 1 27 127 12 658 21 16 37 ZAG M 85% 35 28 39 96 73 1 11 42 17 18 ZCR M 80% 84 11 39160 22 38 14 57 20 18 ZIJ M 72% 6 33 31 320 29 13 25 34 3 13 BQL (BelowQuantization Limit) TBD (To Be Determined)

Example 3 Characterization of Pooled Hepatocytes

Cryopreserved pooled lots of hepatocytes are prepared and analyzed forpost-thaw viability and enzyme function. The following metabolicactivities are evaluated: COUM, DEX, ECOD, 7-HCG, 7-HCS, MEPH, TEST,TOLB, PHEN, and CZX.

Six lots of pooled hepatocytes, comprising either five-donor pools orten-donor pools, are prepared as described above. Hepatocytes areharvested from individual donors and then cryopreserved as individuallots using liquid nitrogen as freezing agent. Cryopreservation isaccomplished by suspending the hepatocytes into freezer-safe vialscontaining a medium having approximately 10% DMSD and approximately 90%Cryopreservation Medium. The dispensed hepatocytes are then frozen in acontrolled rate freezer until a final temperature of approximately −80°C. is reached.

To form the pooled hepatocyte preparations, individual lots are thawed,and the viable cells are isolated by PERCOLL® gradient centrifugation.Vials of individual donor cryopreserved hepatocytes were thawed in a 37°C. waterbath (perhaps it would be better to give a range such as 30-40°C. waterbath?) for 60-90 seconds. The thawed cells are decanted into 37°C. media containing 30% Isotonic PERCOLL® and 70% CP-2 media. The cellsuspension is centrifuged at 100 g for 20 minutes. The viable cells arerecovered in cryopreservation media and counted. The viable cells arediluted to 20 million cells per mL. A second solution containing 20%DMS4 and 80% cryopreservation media (equal volume to the cell suspensionlisted above) is prepared. The 20% DMSO and 80% cryopreservation mediais slowly added to the cells suspension mixture. The addition takes 5-10minutes. The resulting mixture is 10% DMSO, 90% cryopreservation mediawith cells at 10 million cells per mL. This solution is aliquoted intocryovials at 1.0 mL per vial. The cells are then cryopreserved. Viablecells from individual lots are then pooled to form pooled hepatocytepreparations whose cells have functional assay values within desiredranges.

The pooled lots are then cryopreserved. Table IV below shows the resultsof the post-thaw viability (“% V”) and enzyme function analysis of thepooled lots. As indicated in Table III, pools had an average viabilityof 79% (S.D. ±6%).

TABLE IV Pool % V COUM DEX ECOD 7-HCG 7-HCS MEPH TEST TOLB PHEN CZXMJI^(a) 89 63 28 66 301 44 12 70 61 50 62 YDJ^(a) 72 66 30 80 470 41 1165 31 27 71 APO 79 79 21 55 276 43 2 112 22 26 30 HMB^(b) 76 61 18 70231 46 3 151 23 20 83 IJU^(a) 75 32 19 35 232 44 2 124 32 11 42 RKS^(b)81 84 20 73 336 53 2 131 28 23 55 ^(a)5-donor lots ^(b)10-donor lots

For comparison, Table V below shows summary data for a post-thawviability and enzyme function analysis of eighty-one individual lotsthat are cryopreserved (i.e., subjected to one cycle ofcryopreservation). This data confirms that the lot-to-lot variability ofenzyme function found in individual hepatocyte sources is very high. Thedata confirms the desirability of employing pooled hepatocytepreparations for providing cryopreserved cells that approximate theenzyme function of “average” hepatocytes for a wide variety of enzymes.

TABLE V Summary of Pooled Hepatocyte Lot Data % V COUM DEX ECO 7-HCG7-HCS MEPH TEST TOLB PHEN CZX Avg 79 37 17 51 276 27 8 35 35 15 19 High95 134 69 110 471 110 177 675 82 125 215 Low 62 6 1 31 231 24 2 25 34 313

Example 4

Characterization of the Viability of Pooled Hepatocytes After Thawing

A common use of for cryopreserved hepatocytes is to thaw the hepatocytesand then incubate them with various xenobiotics. For this purpose, it ispreferred that the hepatocytes maintain their viability for at leastseveral hours. To examine the post-thaw viability over time for one lotof pooled cryopreserved hepatocytes, the cells were thawed, aliquotedinto the wells of a 12-well plate, and incubated at 37° C. with 5% CO₂.The viability of the hepatocytes is then measured at time-points for upto six hours. Table VI shows the results of this analysis, wherein, atsix hours, 39% of the hepatocytes remained viable

TABLE VI Post-Thaw Viability Analysis of a Pooled Hepatocyte LotTimepoint % Viability^(a) T = 0 88% 0.5 hrs 79% 1.0 hrs 84% 2.0 hrs 79%3.0 hrs 73% 4.0 hrs 67% 6.0 hrs 69% ^(a)viability determined by TrypanBlue

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth

1. (canceled)
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 9. (canceled)
 10. A method of producing a desired preparation of multi-cryopreserved hepatocytes, said hepatocytes being capable of being frozen and thawed at least two times, and in which greater than 50% of the hepatocytes of said preparation are viable after the final thaw, said method comprising: (A) subjecting hepatocytes that have been frozen and thawed to density gradient fractionation to separate viable hepatocytes from non-viable hepatocytes, (B) recovering the separated viable hepatocytes, and (C) cryopreserving the recovered viable hepatocytes to thereby form said desired preparation of hepatocytes without requiring a density gradient step after thawing the hepatocytes for the second time, wherein the hepatocytes are not plated between the first and second cryopreservations, and wherein greater than 50% of the hepatocytes of said preparation are viable after the final thaw.
 11. The method of claim 10, wherein said density gradient fractionation comprises density centrifugation through polyvinylpyrrolidone-coated colloidal silica particles.
 12. The method of claim 10, wherein said hepatocytes are selected from the group consisting of human hepatocytes, porcine hepatocytes, simian hepatocytes, canine hepatocytes, feline hepatocytes, bovine hepatocytes, equine hepatocytes, ovine hepatocytes and rodent hepatocytes.
 13. The method of claim 12, wherein said hepatocytes are human hepatocytes.
 14. The method of claim 10, wherein said preparation comprises a pooled preparation of hepatocytes of multiple sources.
 15. The method of claim 14, wherein said multiple sources are of the same gender, race, or health state.
 16. The method of claim 14, wherein the hepatocytes of said pooled preparation of hepatocytes provide said pooled preparation with a desired level of a metabolic activity.
 17. The method of claim 16, wherein said metabolic activity is selected from the group consisting of coumarin 7-hydroxylase (COUM), dextromethorphan O-demethylase (DEX), 7-ethoxycourmarin O-deethylase (ECOD), activities responsible for the phase II metabolism of 7-hydroxycoumarin (7-HCG and 7-HCS), mephenyloin 4-hydroxylase (MEPH), testosterone 6(β)-hydroxylase (TEST), tolbutamide 4-hydroxylase (TOLB), phenacetin O-deethylase (PHEN), and chlorzoxazone 6-hydroxylase (CZX).
 18. The method of claim 10, wherein greater than about 70% of the hepatocytes of said preparation are viable.
 19. The method of claim 10, wherein greater than 80% of the hepatocytes of said preparation are viable.
 20. A method of investigating in vitro drug metabolism comprising incubating hepatocytes of a multi-cryopreserved hepatocyte preparation in the presence of a xenobiotic, and determining the metabolic fate of the xenobiotic, or the effect of the xenobiotic on the hepatocytes or on an enzyme or metabolic activity thereof, wherein the hepatocytes have been frozen and thawed at least two times, and wherein greater than 50% of the hepatocytes of said preparation are viable after a final thaw and without requiring a density gradient step after thawing the hepatocytes for the second time, wherein the hepatocytes are not plated between the first and second cryopreservations.
 21. The method of claim 20, wherein greater than about 70% of the hepatocytes of said preparation are viable.
 22. The method of claim 14, wherein said multiple sources are of different gender, race or health state.
 23. The method of claim 16, wherein said multiple sources are selected based upon metabolic activity, and wherein the pooled preparation exhibits a desired level of one or more metabolic activities. 